TOXICITY ASSAYS FOR BUPIVACAINE:HYDROXYPROPYLBETA-CYCLODEXTRIN INCLUSION COMPLEX: CELL VIABILITY AND NITRIC OXIDE PRODUCTION
D. R. Araújo; T. H. N. Simões; M.S. Corrêa and E. de Paula.
Departamento de Bioquímica, Instituto de Biologia/Unicamp, C.P. 6109, CEP 13083-970, Campinas-SP. E-mail: draraujo2003@unicamp.br
Considerable interest has focused on the use of short-term in vitro cytotoxicity tests with cultured cells for the evaluation of the acute toxicity of chemical agents. Bupivacaine (BVC) is an amino-amide local anesthetic (LA) widely used for regional nerve blockade. Nevertheless, local and systemic toxicity of BVC (to the central nervous and cardiovascular systems) restricts its safe clinical use. Nitric oxide (NO), a neurotransmitter and endothelium-derivative relaxing factor, plays an essential role in the regulation of brain and cardiovascular function; the role of NO in seizure activity and cardiac arrhythmias induced by LA is of great interest. The present study aimed to evaluate the in vitro toxicity of an inclusion complex, prepared with racemic BVC and hydroxypropylbeta-cyclodextrin (HPb-CD) by reporting its effect on cell viability and NO production. Inclusion complexes were obtained by mixing appropriate amounts of HPb-CD and BVC to a final 1:1 molar ratio. Cytotoxic assays were performed using Balb/c fibroblasts, 3T3 cells, cultured in DMEM (supplemented with 10% fetal bovine serum, pH 7.2-7.4, under humidified atmosphere at 37 ºC and 5% CO2); the cells were seeded (2 x 104 cells/well) in 96-wells tissue culture plates and cultured for 48 h. After that the cells were incubated for 2 h with the vehicle (HPb-CD) or with bupivacaine (free BVC or BVCHPb-CD) formulations. Cell viability was determined by measuring the amount of tetrazolium (MTT) converted to formazan and also by neutral red (NR) retention. NO contents were determined by nitrite detection. MTT reduction and NR retention tests showed that free BVC (0.5; 0.25 and 0.125%), reduced the cell viability up to 50%. BVCHPb-CD, in a different manner, did not affect cell viability up to 2 hours after treatment, an effect similar to that found with HPb-CD (p<0.01 and p<0.05, respectively), in comparison to free BVC. The NO content of the 3T3 cells was increased after treatment with BVCHPb-CD (p<0.001 when compared to free BVC p<0.01), showing that the amount of BVC available in the cell after complexation was greater than before. Cell culture assays point BVCHPb-CD as a less toxic formulation than BVC in solution, and show the relationship between the cellular concentration of bupivacaine and free radicals, such as NO, generation. Supported by FAEPEX/UNICAMP
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